Influenza (flu) is a contagious respiratory illness caused by flu viruses, leading to about 36,000 deaths every year in the United States alone, with the potential for at least a ten fold increase in epidemic and pandemic scenarios. During attachment of flu viruses to epithelial cells, hemagglutinin, one of its surface proteins, binds to sialic acid residues, initiating a series of events leading to activation of PKC, which in turn, down-regulates the activity of amiloride sensitive epithelial Na+ channels (ENaC) of tracheal and alveolar cells. It has been thought that these events are responsible for flu-induced rhinorrhea and life-threatening alveolar edema in humans. However, events occurring during the attachment of influenza virus to epithelial cells are likely to be transient and relatively few cells will be initially affected. We propose that M2, a transmembrane protein that plays a critical role in viral replication, enhances intracellular production of reactive oxygen-nitrogen species (RONS) which (i) oxidize and nitrate ENaC; and (ii) activate PKC(. Both processes enhance ENaC ubiquitination and subsequent destruction by the proteasome or lysosome systems. These hypotheses will be tested by completing the following comprehensive in vitro and in vivo studies listed in four specific aims: (1) Identify regions and specific amino acids of the influenza strain A/Udorn/72 M2 proton (H+) channel responsible for ENaC down-regulation in Xenopus oocytes microinjected with 1-,2-, and 3-ENaC. (2) Identify the mechanisms by which M2 decreases ENaC protein levels and function. We propose that M2 enhances intracellular production of reactive oxygen-nitrogen species (RONS) which (i) oxidize and nitrate ENaC; and (ii) activate PKC(. Both processes enhance ENaC ubiquitination and subsequent destruction by the proteasome or lysosome systems (3) Identify the mechanisms by which M2 inhibits amiloride sensitive Na+ currents in human airway (H441) and rat alveolar type II (rATII) cells, expressing native ENaC and (4) Establish the contribution of M2 in the inhibition of lung fluid clearance of mice infected by replicating flu viruses and identify the mechanisms involved. The results of our studies may provide the rational basis for the development of new therapeutic strategies, against a highly conserved region of the viral genome to knockdown M2 expression, and thus broadly and effectively decrease flu induced pulmonary edema and rhinorrhea. Due to the public health impact of influenza, there is a strong need to investigate and develop therapies that address the host response to viral infection, which may contribute to the morbidity and mortality of pathogenic respiratory viruses. PUBLIC HEALTH RELEVANCE: The results of our studies will provide the rational basis for the development of new therapeutic strategies, such as administration of agents to decrease M2 expression, and thus broadly and effectively decrease the flu-induced rhinorrhea, alveolar edema and hypoxemia. Due to the public health impact of influenza, there is a strong need to investigate and develop therapies that address the host response to viral infection, which may contribute to the morbidity and mortality of pathogenic respiratory viruses.